Multipotent neural stem cells from peripheral tissues and uses thereof

ABSTRACT

This invention relates to multipotent neural stem cells, purified from the peripheral nervous system of mammals, capable of differentiating into neural and non-neural cell types. These stem cells provide an accessible source for autologous transplantation into CNS, PNS, and other damaged tissues.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of and claims priorityto PCT application CA01/00047 filed Jan. 24, 2001 and to U.S.application Ser. No. 09/670,049, filed Sep. 25, 2000, which applicationis a continuation-in-part of application Ser. No. 09/490,422, filed Jan.24, 2000, each of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to multipotent stem cells (MSCs)purified from peripheral tissues including peripheral tissues containingsensory receptors such as skin, olfactory epithelium, mucosa, andtongue. The invention also relates to cells differentiated from thesemultipotent stem cells. The invention includes pharmaceuticalcompositions and uses of either the multipotent stem cells or thedifferentiated cells derived from such stem cells. Additionally,business methods based on the multipotent stem cells or thedifferentiated cells are contemplated.

[0003] There are a number of diseases of the central nervous system(“CNS”) which have a devastating effect on patients. These diseases aredebilitating, often incurable, and include, for example, Alzheimer'sdisease, Huntington's disease, Parkinson's disease, and MultipleSclerosis.

[0004] By way of example, Parkinson's disease is a progressivedegenerative disorder of unknown cause. In healthy brain tissue,dopaminergic neurons extend from the substantia nigra of the brain intothe neighboring striatum. In Parkinson's disease, these dopaminergicneurons die.

[0005] There are a number of methods to treat Parkinson's disease. Onemethod is to treat humans having Parkinson's disease with L-DOPA. Asecond method is to transplant cells into the substantia nigra orstriatum. Transplanted cells replace endogenous cells that are lost as aconsequence of disease progression. An animal model of Parkinson'sdisease is an MPTP-treated non-human primate. The MPTP-treated animalshave been transplanted with dopamine-rich embryonic neurons with somesuccess.

[0006] To date, the cells used for neural transplant have been collectedfrom the developing brains of aborted fetuses. Aside from the ethicalconsiderations, the method from a practical standpoint is unlikely toprovide a sufficient amount of neural tissue to meet the demands. Thus,another source of cells for transplantation is desirable.

[0007] Stem cells are undifferentiated cells that exist in many tissuesof embryos and adult organisms. In embryos, blastocyst stem cells arethe source of cells which differentiate to form the specialized tissuesand organs of the developing fetus. In adults, specialized stem cells inindividual tissues are the source of new cells, replacing cells lostthrough cell death due to natural attrition, disease, or injury. Stemcells may be used as substrates for producing healthy tissue where adisease, disorder, or abnormal physical state has destroyed or damagednormal tissue.

[0008] Weiss et al., 1996 summarizes the five defining characteristicsof stem cells as the ability to:

[0009] Proliferate: Stem cells are capable of dividing to producedaughter cells.

[0010] Exhibit self-maintenance or renewal over the lifetime of theorganism: Stem cells are capable of reproducing by dividingsymmetrically or asymmetrically to produce new stem cells. Symmetricdivision occurs when one stem cell divides into two daughter stem cells.Asymmetric division occurs when one stem cell forms one new stem celland one progenitor cell. Symmetric division is a source of renewal ofstem cells. This permits stem cells to maintain a consistent level ofstem cells in an embryo or adult mammal.

[0011] Generate large number of progeny: Stem cells may produce a largenumber of progeny through the transient amplification of a population ofprogenitor cells.

[0012] Retain their multilineage potential over time: Stem cells are theultimate source of differentiated tissue cells, so they retain theirability to produce multiple types of progenitor cells, which will inturn develop into specialized tissue cells.

[0013] Generate new cells in response to injury or disease: This isessential in tissues which have a high turnover rate or which are morelikely to be subject to injury or disease, such as the epithelium ofblood cells.

[0014] Thus, the key features of stem cells are that they aremultipotential cells which are capable of long-term self-renewal overthe lifetime of a mammal.

[0015] MSCs may be used as a source of cells for transplantation. Thestem cells may themselves be transplanted or, alternatively, they may beinduced to produce differentiated cells (e.g., neurons,oligodendrocytes, Schwann cells, or astrocytes) for transplantation.Transplanted stem cells may also be used to express therapeuticmolecules, such as growth factors, cytokines, anti-apoptotic proteins,and the like. Thus, stem cells are a potential source of cells foralternative treatments of diseases involving loss of cells or tissues.

[0016] The safest type of tissue graft (using stem cells or otherwise)is one that comes from self (an autologous tissue source). Autologoustissue sources are widely used in procedures such as bone transplantsand skin transplants because a source of healthy tissue is readilyaccessible for transplant to a damaged tissue site. In brain diseases,such as Parkinson's disease, healthy dopaminergic neuronal brain tissuemay exist at other sites in the brain, but attempts to transplant theseneurons may harm the site where the healthy neurons originate.Multipotent stem cells that can be differentiated into dopaminergicneurons may be available at other sites from which they may betransplanted, but the CNS, particularly the brain, is physicallydifficult to access.

[0017] In several tissues, stem cells have been purified andcharacterized. For example, neural stem cells have been purified fromthe mammalian forebrain (Reynolds and Weiss, Science 255:1707-1710,1992) and these cells were shown to be capable of differentiating intoneurons, astrocytes, and oligodendrocytes. PCT publications WO 93/01275,WO 94/16718, WO 94/10292 and WO 94/09119 describe uses for these cells.It could be impractical or impossible, however, to first access brain orother CNS tissue for biopsy and then again for transplant in patientswith weakened health. It would be very useful if there were accessiblestem cells capable of differentiating into CNS cell types, such asdopaminergic neurons; such cells would be a source of cells forautologous transplants.

[0018] Thus, there is a clear need to develop methods for identifyingfrom accessible tissues multipotent stem cells that can act as a sourceof cells that are transplantable to the CNS, PNS, or other tissues invivo in order to replace damaged or diseased tissue.

SUMMARY OF THE INVENTION

[0019] One aspect of the present invention relates to preparations ofpurified multipotent stem cells that are obtained from peripheral tissueof mammals, preferably from postnatal mammals such as juvenile and adultmammals. We have identified epithelial tissues, such as skin, asconvenient sources of multipotent stem cells, and provide methods forthe purification of epithelial-derived MSCs, thus simplifying theharvesting of cells for transplantation relative to previous methods.The MSCs possess desirable features in that they are multipotent andself-renewing. The cells can be repeatedly passaged and can bedifferentiated into numerous cell types of the body includingderivatives of ectodermal and mesodermal tissue. The MSCs of thisinvention are positive for nestin protein, an immunological marker ofstem cells and progenitor cells, as well as fibronectin protein, but arenegative for vimentin or cytokeratin when assayed byimmunohistochemistry. Moreover, the MSCs of the present invention growas non-adherent clusters when cultured by the methods herein disclosed,and one of skill in the art will readily recognize that such cells willgrow as non-adherent clusters when cultured on a variety of substratumincluding but not limited to uncoated plastic or plastic coated with aneutral substrate such as gelatin or agar. In certain embodiments, theMSCs of this invention are negative for the neural crest stem cellmarker p75. These characteristics distinguish the cells of thisinvention from other stem cells, including mesenchymal stem cells andneural crest stem cells.

[0020] In certain embodiments, the cells are capable of differentiatingas dopaminergic neurons, and thus are a useful source of dopaminergicneurons for homotypic grafts into Parkinson's Disease patients. The MSCshave also been demonstrated to make numerous mesodermal derivativesincluding smooth muscle cells, adipocytes, bone, and are expected to becapable of producing other mesodermal and endodermal cell typesincluding cardiac muscle cells, pancreatic islet cells (e.g., alpha,beta, phi, delta cells), hematopoietic cells, hepatocytes, and the like.The subject cells may also be used for autologous or heterologoustransplants to treat, for example, other neurodegenerative diseases,disorders, or abnormal physical states.

[0021] Accordingly, in a first aspect, the invention features MSCssubstantially purified from a peripheral tissue of a postnatal mammal.In preferred embodiments, the peripheral tissue is an epithelial tissueincluding skin or mucosal tissue. In a second embodiment, the peripheraltissue is derived from the tongue. The postnatal mammal may be either ajuvenile or adult mammal.

[0022] In certain embodiments, the invention features a cell that is theprogeny of a MSC substantially purified from a peripheral tissue of apostnatal mammal. The cell may be a mitotic cell or a differentiatedcell (e.g., a neuron, an astrocyte, an oligodendrocyte, a Schwann cell,or a non-neural cell). Preferred neurons include neurons expressing oneor more of the following neurotransmitters: dopamine, GABA, glycine,acetylcholine, glutamate, and serotonin. Preferred non-neural cellsinclude cardiac muscle cells, pancreatic cells (e.g., islet cells(alpha, beta, phi and delta cells,)), exocrine cells, endocrine cells,chondrocytes, osteocytes, skeletal muscle cells, smooth muscle cells,hepatocytes, hematopoietic cells, and adipocytes. In a preferredembodiment, the differentiated cells are substantially purified.

[0023] In a second aspect, the invention features a population of atleast ten cells, wherein at least 30% of the cells are MSCssubstantially purified from a peripheral tissue of a postnatal mammal orprogeny of the MSCs.

[0024] Preferably, at least 50% of the cells are MSCs substantiallypurified from the peripheral tissue or progeny of the MSCs. Morepreferably, at least 75% of the cells are MSCs substantially purifiedfrom the peripheral tissue or progeny of the MSCs. Most preferably, atleast 90%, 95%, or even 100% of the cells are MSCs substantiallypurified from the peripheral tissue or progeny of the MSCs. The MSCs maybe cultured for extended periods of time. Thus, the population of cellsmay have been in culture for at least thirty days, sixty days, ninetydays, or longer (e.g., one year or more). Preferably, the population isat least twenty cells, and may be more than fifty cells, a thousandcells, or even a million cells or more.

[0025] In a third aspect, the invention features preparations of atleast ten cells, and more preferably at least 10⁴, 10⁵, 10⁶ or even 10⁷cells, having less than 25% lineage committed cells. Preferably, lessthan 20% of the cells are lineage committed cells. More preferably, lessthan 15% of the cells are lineage committed cells. Most preferably, lessthan 10%, 5%, or even 0% of the cells are lineage committed cells. Ingeneral, any cell feeder layer upon which the cells of the invention arecultured would not be considered in such a calculation.

[0026] In a fourth aspect, the invention features a pharmaceuticalcomposition including (i) a mitotic or differentiated cell that is theprogeny of a MSC substantially purified from a peripheral tissue of apostnatal mammal, and (ii) a pharmaceutically acceptable carrier,auxiliary or excipient.

[0027] In a fifth, related aspect, the invention features apharmaceutical composition including (i) a MSC substantially purifiedfrom a peripheral tissue of a postnatal mammal, and (ii) apharmaceutically acceptable carrier, auxiliary or excipient.

[0028] Preferably, the composition of the fourth or fifth aspectincludes a population of cells, wherein at least 30%, 50%, 75%, 90%,95%, or even 100% of the cells are MSCs substantially purified from theperipheral tissue or progeny of the MSCs. The composition may includeone or more types of cells selected from a group consisting of MSCs, orneurons, oligodendrocytes, Schwann cells, astrocytes, adipocytes, smoothmuscle cells, cardiomyocytes, chondrocytes, osteocytes, skeletal musclecells, hepatocytes, hematopoietic cells, exocrine cells, endocrine cellsand alpha, beta, phi and delta cells, which are progeny of MSCs.

[0029] In a sixth aspect, the invention features a method of producing apopulation of at least ten cells, wherein at least 30% of the cells areMSCs substantially purified from a peripheral tissue of a postnatalmammal or progeny of the MSCs: (a) providing the peripheral tissue fromthe mammal; (b) culturing the tissue under conditions in which MSCsproliferate and in which at least 25% of the cells that are not MSCsdie; and (c) continuing culture step (b) until at least 30% of the cellsare MSCs or progeny of the MSCs.

[0030] In a seventh aspect, the invention features another method ofproducing a population of at least ten cells, wherein at least 30% ofthe cells are MSCs substantially purified from skin tissue of apostnatal mammal or progeny of the MSCs, the method including: (a)providing the skin tissue from the mammal; (b) culturing the tissueunder conditions in which MSCs proliferate and in which at least 25% ofthe cells that are not MSCs die; (c) separating the MSCs from cells thatare not MSCs based on the tendency of MSCs to form non-adherentclusters; and (d) repeating steps (b) and (c) until at least 30% of thecells are MSCs or progeny of the MSCs.

[0031] Suitable culture conditions for step (b) of the sixth and seventhaspects are preferably as follows: (i) triturating or otherwiseseparating tissue into single cells or cell clusters and placing intoculture medium; (ii) culturing the cells in culture medium and underconditions (e.g., DMEM: Ham's F-12 medium containing B-27 supplement,antibacterial and antifungal agents, 5-100 ng/ml bFGF, and 2-100 ng/mlEGF) that allows for the proliferation of MSCs but does not promote, tothe same extent, proliferation of cells that are not MSCs; and (iii)culturing the separated tissue for three to ten days, during which timethe MSCs proliferate in suspension and form non-adherent clusters butnon-MSCs do not proliferate in suspension (these cells either attach tothe plastic or they die). Preferably, at least 50% of the cells insuspension surviving after the period in culture are MSCs or progeny ofthe MSCs, more preferably, at least 75% of the cells are MSCs or progenyof the MSCs, and, most preferably, at least 90% or even 95% of thesurviving cells are MSCs or progeny of the MSCs. In preferredembodiments tissue is separated mechanically.

[0032] In an eighth aspect, the invention features a method of treatinga patient having a disease associated with cell loss. In one embodiment,the method includes the step of transplanting the multipotent stem cellsof the invention into the region of the patient in which there is cellloss. Preferably, prior to the transplanting step, the method includesthe steps of providing a culture of peripheral tissue and isolating amultipotent stem cell from the peripheral tissue. The tissue may bederived from the same patient (autologous) or from either a geneticallyrelated or unrelated individual. After transplanation, the method mayfurther include the step of differentiating (or allowing thedifferentiation of) the MSCs into a desired cell type to replace thecells that were lost. Preferably, the region is a region of the CNS orPNS, but can also be cardiac tissue, pancreatic tissue, or any othertissue in which cell transplantation therapy is possible. In a secondembodiment, the method includes the step of delivering the stem cells tothe site of cell damage via the bloodstream, wherein the stem cells hometo the site of cell damage. In a third embodiment, the method fortreating a patient includes the transplantation of the differentiatedcells which are the progeny of the stem cells of this invention.

[0033] In a ninth aspect, the invention features a kit including MSCssubstantially purified from peripheral tissue of a postnatal mammal, ora mitotic or differentiated cell that is the progeny of the MSC,preferably wherein the peripheral tissue from which the MSC is purifiedincludes a sensory receptor. Preferably, the kit includes a populationof cells, wherein at least 30%, 50%, 75%, 90%, or even 95% of the cellsare MSCs substantially purified from the peripheral tissue or progeny ofthe MSCs.

[0034] In a tenth aspect, the invention features a kit for purifyingMSCs from peripheral tissue. The kit includes media or media componentsthat allow for the substantial purification of MSCs of the presentinvention. The kit may also include media or media components that allowfor the differentiation of the MSCs into the desired cell type(s).Preferably, the kit also includes instructions for its use.

[0035] In one preferred embodiment of each of the foregoing aspects ofthe invention, the peripheral tissue is skin tissue. In anotherpreferred embodiment, the peripheral tissue is tongue tissue, hairfollicles, sweat glands, or sebaceous glands. In another preferredembodiment of each of the foregoing aspects of the invention, the stemcells are negative for p75.

[0036] The peripheral tissue can be from a newborn mammal, a juvenilemammal, or an adult mammal. Preferred mammals include, for example,humans, non-human primates, mice, pigs, and rats. The MSCs can bederived from peripheral tissue of any individual, including onesuffering from a disease or from an individual immunologicallycompatible to an individual suffering from a disease. In a preferredembodiment, the cells, or progeny of the cells, are transplanted intothe CNS or PNS of an individual having a neurodegenerative disease andthe individual is the same individual from whom the MSCs were purified.Following transplantation, the cells can differentiate into cells thatare lacking or non-functional in the disease.

[0037] Preferably, the MSCs are positive for nestin and fibronectinprotein and may also express glutamic acid decarboxylase, but arenegative for vimentin and cytokeratin protein. The MSCs of the presentinvention can, under appropriate conditions, differentiate into neurons,astrocytes, Schwann cells, oligodendrocytes, and/or non-neural cells(e.g., cardiac cells, pancreatic cells, smooth muscle cells, adipocytes,hepatocytes, etc.). In a preferred embodiment, the differentiatedneurons are dopaminergic neurons.

[0038] MSCs can be stably or transiently transfected with a heterologousgene (e.g., one encoding a therapeutic protein, such as a protein whichenhances cell divisions or prevents apoptosis of the transformed cell orother cells in the patient, or a cell fate-determining protein). Inpreferred embodiments, transfection of the heterologous gene isadenoviral mediated. In another preferred embodiment, transfectionoccurs using standard protocols for transfection in cell cultureincluding lipofectamine mediated transfection or electroporation.

[0039] In an eleventh aspect, the invention features preparations ofstem cells and their differentiated progeny preserved for subsequentretrieval. In one preferred embodiment, the preserved cells areformulated in a pharmaceutically acceptable carrier. In anotherembodiment, the stem cells or differentiated progeny are preserved usingcryogenic methods.

[0040] In a twelfth aspect, the invention features a method forconducting a regenerative medicine business. In one embodiment, themethod comprises accepting and cataloging tissue samples from a client,culturing the cells from said sample to expand the multipotent stemcells, preserving such cells and storing them for later retrieval. In asecond embodiment, the method comprises accepting and cataloging tissuesamples from a client, culturing the cells from said sample to expandthe multipotent stem cells, and differentiating the stem cell. Both ofthese embodiments also contemplate a billing system for billing theclient or an insurance provider.

[0041] In a thirteenth aspect, the invention features a method forconducting a stem cell business comprising identifying factors whichinfluence the proliferation, differentiation, or survival of themultipotent stem cells of the invention. Such factors include smallorganic molecules and extracellular protein. In a preferred embodiment,the identified agents could be profiled and assessed for safety andefficacy in animals. In another preferred embodiment, the identifiedagents are formulated as a pharmaceutical preparation. Thispharmaceutical preparation can be manufactured, marketed, anddistributed for sale.

[0042] In a fourteenth aspect, the invention includes a method forconducting a drug discovery business comprising identifying factorswhich influence the proliferation, differentiation, or survival of themultipotent stem cells of the invention, and licensing the rights forfurther development.

[0043] For convenience, certain terms employed in the specification,examples, and appended claims are collected here.

[0044] By “multipotential stem cell” is meant a cell that (i) has thepotential of differentiating into at least two cell types selected froma neuron, an astrocyte, and an oligodendrocyte, and (ii) exhibitsself-renewal, meaning that at a cell division, at least one of the twodaughter cells will also be a stem cell. The non-stem cell progeny of asingle MSC are capable of differentiating into neurons, astrocytes,Schwann cells, and oligodendrocytes. Hence, the stem cell is“multipotent” because its progeny have multiple differentiativepathways. The MSC also has the potential to differentiate as anothernon-neuronal cell type (e.g., a skin cell, a hematopoietic cell, asmooth muscle cell, a cardiac muscle cell, a skeletal muscle cell, or apancreatic cell).

[0045] By a “population of cells” is meant a collection of at least tencells. Preferably, the population consists of at least twenty cells,more preferably at least one hundred cells, and most preferably at leastone thousand or even one million cells. Because the MSCs of the presentinvention exhibit a capacity for self-renewal, they can be expanded inculture to produce populations of even billions of cells.

[0046] By “substantially purified” is meant that the desired cells(e.g., MSCs) are enriched by at least 30%, more preferably by at least50%, even more preferably by at least 75%, and most preferably by atleast 90% or even 95%.

[0047] By “therapeutic protein” is meant a protein that improves ormaintains the health of the cell expressing the protein or of a cellthat is in proximity to the expressing cell. Examples of therapeuticproteins include, without limitation, growth factors (NGF, BDNF, NT-3,NT-4/5, HGF, TGF-β family members, PDGF, GDNF, FGF, EGF family members,IGF, insulin, BMPs, Wnts, hedgehogs, and heregulins) cytokines (LIF,CNTF, TNFμ interleukins, and gamma-interferon), and anti-apoptoticproteins (IAP proteins, Bcl-2 proteins, Bcl-X_(L), Trk receptors, Akt,PI3 kinase, Gab, Mek, E1B55K, Raf, Ras, PKC, PLC, FRS2, rAPs/SH2B, andNp73).

[0048] By “peripheral tissue” is meant a tissue that is not derived fromneuroectoderm, for example peripheral tissue containing sensoryreceptors, and specifically includes olfactory epithelium, tongue, skin(including dermis and/or epidermis), and mucosal layers of the body(e.g., mouth, reproductive system).

[0049] By “epithelia” and “epithelium” in meant the cellular covering ofinternal and external body surfaces (cutaneous, mucous and serous),including the glands and other structures derived therefrom, e.g.,corneal, esophegeal, epidermal, and hair follicle epithelial cells.Other exemplary epithelial tissue includes: olfactory epithelium, whichis the pseudostratified epithelium lining the olfactory region of thenasal cavity, and containing the receptors for the sense of smell;glandular epithelium, which refers to epithelium composed of secretingcells; squamous epithelium, which refers to epithelium composed offlattened plate-like cells. The term epithelium can also refer totransitional epithelium, that which is characteristically found lininghollow organs that are subject to great mechanical change due tocontraction and distention, e.g. tissue which represents a transitionbetween stratified squamous and columnar epithelium. The term“epithelialization” refers to healing by the growth of epithelial tissueover a denuded surface.

[0050] By “skin” is meant the outer protective covering of the body,consisting of the corium and the epidermis, and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” may beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used.

[0051] By “epidermis” is meant the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers: basal layer composed of columnar cellsarranged perpendicularly; prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, conifiednon-nucleated cells. In the epidermis of the general body surface, theclear layer is usually absent. An “epidermoid” is a cell or tissueresembling the epidermis, but may also be used to refer to any tumoroccurring in a noncutaneous site and formed by inclusion of epidermalelements.

[0052] By “ectoderm” is meant the outermost of the three primitive germlayers of the embryo; from which are derived the epidermis and epidermaltissues such as the nails, hair and glands of the skin, the nervoussystem, external sense organs and mucous membrane of the mouth and anus.

[0053] By “mesoderm” is meant the middle of the three primitive germlayers of the embryo; from which are derived the heart, kidney, skeletalmuscle, bone, blood, endothelial lining of blood vessels, adiposetissue, and the urogenital system.

[0054] By “endoderm” is meant the innermost of the three primitive germlayers of the embryo; from which are derived the lungs, trachea,pharynx, thyroid, pharyngeal pouch derivatives, and the organs of thegut including the stomach, small intestines, large intestines, pancreas,liver, gall bladder, appendix, esophagus, rectum, anus, and urinarybladder.

[0055] By “differentiation” is meant the formation of cells expressingmarkers known to be associated with cells that are more specialized andcloser to becoming terminally differentiated cells incapable of furtherdivision or differentiation.

[0056] By “lineage committed cell” is meant a progenitor cell that is nolonger pluripotent but has been induced to differentiate into a specificcell type, e.g., a dopaminergic neuron.

[0057] By “proliferation” is meant an increase in cell number.

[0058] By “non-adherent clusters” is meant that the cells of theinvention are able to adhere to each other and form clusters whichincrease in size as the cells proliferate, but these cells do not adhereto the substratum and grow in suspension, wherein the substratum isuncoated tissue culture plastic or a culture vessel coated with aneutral coating such as agar or gelatin.

[0059] By “dissociating a sample” is meant to separate tissue intoeither single cells, smaller cell clusters, or smaller pieces of tissue.

[0060] By “postnatal” is meant an animal that has been born at term.

[0061] By “a disease characterized by failure of a cell type” is meantone in which the disease phenotype is the result of loss of cells ofthat cell type or the loss of function of cells of that cell type.

[0062] By “autologous transplant” is meant that the transplantedmaterial (e.g., MSCs or the progeny or differentiated cells thereof) isderived from the same individual.

[0063] By “nucleic acid” is meant polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

[0064] By “gene” is meant a nucleic acid comprising an open readingframe encoding a polypeptide, including both exon and (optionally)intron sequences.

[0065] By “transfection” is meant the introduction of a nucleic acid,e.g., an expression vector, into a recipient cell by nucleicacid-mediated gene transfer.

[0066] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of preferred vector is an episome, i.e., a nucleicacid capable of extra-chromosomal replication. Preferred vectors arethose capable of autonomous replication and/expression of nucleic acidsto which they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

[0067] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. It will also be understood that the recombinant gene can beunder the control of transcriptional regulatory sequences which are thesame or which are different from those sequences which controltranscription of the naturally-occurring gene.

[0068] By “tissue-specific promoter” is meant a DNA sequence that servesas a promoter, i.e., regulates expression of a selected DNA sequenceoperably linked to the promoter, and which effects expression of theselected DNA sequence in specific cells of a tissue, such as cells ofneuronal or hematopoietic origin. The term also covers so-called “leaky”promoters, which regulate expression of a selected DNA primarily in onetissue, but can cause at least low level expression in other tissues aswell.

[0069] Other features and advantages of the present invention willbecome apparent from the following detailed description and the claims.It will be understood, however, that the detailed description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of example only, and various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIGS. 1A-1G are photographs showing that mouse skin-derived MSCsare nestin-positive and are capable of differentiating into neurons,glia, and smooth muscle cells.

[0071]FIG. 2 is a series of photographs showing that neonate and adultmouse skin-derived MSCs express both nestin (middle row) and fibronectinprotein(bottom row).

[0072]FIG. 3A is a series of photographs showing western blot analysisfor nestin, neurofilament M (NF-M) and GFAP in cells differentiated fromneonate and adult mouse skin-derived MSCs.

[0073]FIG. 3B is a series of photographs showing that human skin-derivedMSCs express nestin.

[0074]FIG. 3C is a series of photographs showing that a subset ofmorphologically complex cells expressed nestin and βtubulin, a profiletypical of newly-born neurons.

[0075]FIG. 3D is a series of photographs showing that GFP positive cellsare also positive for neuron-specific enolase.

[0076]FIG. 4A is a photograph showing the expression of A2B5, a markerfor oligodendrocyte precursors, on undifferentiated mouse skin-derivedMSCs.

[0077]FIG. 4B is a photograph showing the expression of theoligodendrocyte marker galactocerebroside (GalC) on cells differentiatedfrom mouse skin-derived MSCs.

[0078]FIG. 5 is a series of photographs showing that the fate of mouseskin-derived MSCs can be manipulated by controlling plating conditions.

[0079]FIG. 6 is a series of photographs showing that neonate and adultmouse skin-derived MSCs can differentiate as adipocytes.

[0080]FIGS. 7A and 7B are photographs showing that nestin-positive,fibronectin-positive MSCs can be derived from mouse dermis.

[0081]FIGS. 8A and 8B are photographs showing that individual MSCs aremultipotent. Clones derived from single cells contained NF-M-positivecells (arrowheads) and CNPase-positive cells (arrows) (FIG. 3A). In FIG.3B, arrowheads indicate cells that only express GFAP, while arrowsindicate cells expressing both GFAP and CNPase.

[0082]FIGS. 9A and 9B are photographs of western blot analysis of cellsdifferentiated from mouse skin-derived MSCs (FIG. 9A) or of MSCsthemselves (FIG. 9B).

[0083]FIG. 10 is a series of photographs showing the effect of variouspharmacological agents on mouse skin-derived MSCs.

[0084] FIGS. 11A-11E are photographs of immunoprocessed sections of ratbrains into which mouse skin-derived MSCs were transplanted.

DETAILED DESCRIPTION OF THE INVENTION

[0085] We have substantially purified multipotent stem cells (MSCs) fromperipheral tissues of mammals, including skin, olfactory epithelium, andtongue. These cells proliferate in culture, so that large numbers ofstem cells can be generated. These cells can be induced todifferentiate, for example, into neurons, astrocytes, and/oroligodendrocytes by altering the culture conditions. They can also beinduced to differentiate into non-neural cells such as smooth musclecells, bone and adipocytes. The substantially purified neural stem cellsare thus useful for generating cells for use, for example, in autologoustransplants for the treatment of degenerative disorders or trauma (e.g.,spinal cord injury). In one example, MSCs may be differentiated intodopaminergic neurons and implanted in the substantia nigra or striatumof a Parkinson's disease patient. In a second example, the cells may beused to generate oligodendrocytes for use in autologous transplants forthe treatment of multiple sclerosis. In a third example, the MSCs may beused to generate Schwann cells for treatment of spinal cord injury,cardiac cells for the treatment of heart disease, or pancreatic isletcells for the treatment of diabetes. In still another example, MSCs maybe used to replace cells damaged or lost to bacterial or viralinfection, or those lost to traumatic injuries such as burns, fractures,and lacerations. If desired, in any of the foregoing examples, the cellsmay be genetically modified to express, for example, a growth factor, ananti-apoptotic protein, or another therapeutic protein.

[0086] The MSCs display some similarities to stem cells derived frommammalian forebrain, but also possess some distinctive differences. Inparticular, when the MSCs of the present invention differentiate in thepresence of serum, about 5-20% of the differentiated cells expressneuronal markers, whereas differentiated forebrain stem cells generateonly a small percentage of neurons. Moreover, significant numbers ofdopaminergic neurons are found in differentiated cultures of MSCs of thepresent invention, whereas such neurons have not been observed incultures of forebrain stem cells differentiated in serum.

[0087] Cell Therapy

[0088] The multipotent stem cells of this invention may be used toprepare pharmaceutical compositions that can be administered to humansor animals for cell therapy. The cells may be undifferentiated ordifferentiated prior to administration. Dosages to be administereddepend on patient needs, on the desired effect, and on the chosen routeof administration.

[0089] The invention also features the use of the cells of thisinvention to introduce therapeutic compounds into the diseased, damaged,or physically abnormal CNS, PNS, or other tissue. The MSCs thus act as avector to transport the compound. In order to allow for expression ofthe therapeutic compound, suitable regulatory elements may be derivedfrom a variety of sources, and may be readily selected by one withordinary skill in the art. Examples of regulatory elements include atranscriptional promoter and enhancer or RNA polymerase bindingsequence, and a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the vector employed, othergenetic elements, such as selectable markers, may be incorporated intothe recombinant molecule. The recombinant molecule may be introducedinto the stem cells or the cells differentiated from the stem cellsusing in vitro delivery vehicles such as retroviral vectors, adenoviralvectors, DNA virus vectors and liposomes. They may also be introducedinto such cells in vivo using physical techniques such as microinjectionand electroporation or chemical methods such as incorporation of DNAinto liposomes. Such standard methods can be used to either transientlyor stably introduce heterologous recombinant molecules into the cells.The genetically altered cells may be encapsulated in microspheres andimplanted into or in proximity to the diseased or damaged tissue.

[0090] In one embodiment, the MSCs are used for the treatment ofneurological disease. In another aspect the MSCs of the presentinvention may also be used as a source of non-neural cells, for exampleadipocytes, bone and smooth muscle cells. As an example, PCT publicationWO99/16863 describes the differentiation of forebrain MSCs into cells ofthe hematopoietic cell lineage in vivo. The MSCs of the presentinvention appear to be more plastic and thus are highly likely to alsobe capable of differentiating into non-neural cells types, such ashematopoietic cells. Accordingly, the invention features methods oftreating a patient having any disease or disorder characterized by cellloss by administering MSCs of the present invention (or cells derivedfrom these cells) to that patient and allowing the cells todifferentiate to replace the cells lost in the disease or disorder. Forexample, transplantation of MSCs and their progeny provide analternative to bone marrow and hematopoietic stem cell transplantationto treat blood-related disorders. Other uses of the MSCs are describedin Ourednik et al. (Clin. Genet. 56:267-278, 1999), hereby incorporatedby reference. MSCs and their progeny provide, for example, cultures ofadipocytes and smooth muscle cells for study in vitro and fortransplantation. Adipocytes secrete a variety of growth factors that maybe desirable in treating cachexia, muscle wasting, and eating disorders.Smooth muscle cells may be, for example, incorporated into vasculargrafts, intestinal grafts, etc. Transplantation and delivery of MSC'sand their progeny may be at the actual site of cell damage or via theblood stream.

[0091] The following examples describe (i) the derivation of MSCs frompostnatal and adult mouse and rat tissue, (ii) the derivation of MSCsfrom human tissue, (iii) the differentiation of MSCs in vitro to bothectodermal and mesodermal derivatives, (iv) clonal analysisdemonstrating that single MSCs are multipotent, (v) the effects ofpharmacological inhibitors on MSCs, (vi) the transformation of MSCs withexogenous DNA, (vii) the in vivo differentiation of MSCs followingtransplantion.

EXAMPLE 1

[0092] Purification of MSCs from Postnatal Mouse Olfactory Epithelium

[0093] MSCs from mouse olfactory epithelium were purified as describedbelow. Postnatal mice were stunned with a blow to the head and thendecapitated. The heads were sagitally sectioned with a razor blade, andthe olfactory epithelia of about six postnatal (P1-P9) mouse pups werestripped from the conchae, nasal septum, and vomeronasal organs usingwatch-maker forceps. This tissue was placed into 3 mL of medium(DMEM/F-12 3:1) supplemented with 2% B-27 (Gibco, Burlington, Ontario,Canada), 20 ng/mL epidermal growth factor (EGF; Collaborative Research,Bedford, Ma.), 0.1% fungizone, and 0.5 mL/100 mL penicillin/streptomycin(Gibco). Following collection, the epithelia were teased apart withwatchmaker forceps, releasing a large number of single cells and smallcell clusters. The cell suspension was transferred to a 15 mL tube, and7 mL of additional medium was added. The clusters were dissociated intosingle cells by manual titration with a 10 mL plastic pipette and passedthrough a 60 micron filter (Gibco). Typically, dissociated cells fromthe olfactory epithelia from six pups were plated into two 50 mL tissueculture flasks and cultured in a 37 C., 5% CO₂ tissue culture incubator.Two days later, most cells in the cultures were dead or dying. A smallnumber (less than 1% of the initial cell number) of large, phase brightcells were present, however, most of which were attached to the flaskbottom. Over the next two to six days, these cells divided and producedspherical clusters, which became larger over time. At four to five daysin culture, there were approximately 500 clusters of dividing cells perpup used in the original purification. Most of these cell clustersdetached from the flask surface over the next few days. Thesenon-adherent cell clusters continued to grow and fused together tobecome macroscopic, reaching approximately 100 μm in diameter following10 DIV. After 12 DIV, the non-adherent cell clusters became macroscopic,reaching approximately 200 μm or greater in diameter.

[0094] If EGF was not added to the medium, small clusters of dividingcells were still seen by 4 DIV, indicating that the cells themselveswere producing trophic factors in quantities that, in some cases, wassufficient to maintain their proliferation.

[0095] Greater than 95% of the cells in the dividing clusters expressednestin, a marker for stem cells and neural stem cells. Thesenestin-positive cells could be repeatedly passaged, indicating that thecells were stem cells. Six days after purification, the medium (5 mL)was removed from the flasks. This medium contained many clusters ofnon-adherent stem cells that had detached from the flask surface. Thedetached cells clusters were manually triturated with a fire-polishedpipette, thereby dissociating many of the cell clusters into singlecells. The medium containing the cells was then placed in a second flaskwith an additional 15 mL of fresh medium (total volume=20 mL). After afurther six days, one quarter of the medium was removed and thenon-adherent clusters of cells were again triturated and transferred toa new flask with 15 mL fresh medium. These cells have been successfullypassaged more than twenty times without losing their multipotency.

EXAMPLE 2

[0096] Differentiation of Mouse MSCs into Neurons, Astrocytes andOligodendrocytes

[0097] After the cellular clusters of Example 1 had been generated, theycould be differentiated into neurons, astrocytes, and oligodendrocytes.Clusters from cultures 7 to 14 days after purification were plated ontopolylysine coated 35 mm culture dishes or 4 multiwell culture dishes, inDMEM/F12 media containing 2% fetal bovine serum (Hyclone, Logan, Utah)and 2% B-27 (containing no EGF). The medium was changed every three tofour days. Over the next six to nineteen days, cells migrated out of theclusters onto the dish surface. Some of these cells had the morphologyof neurons, astrocytes, or oligodendrocytes. We determined the phenotypeof these cells using the following antibodies: GFAP for astrocytes;neurofilament 160 (NF-160), MAP-2, βIII tubulin, and NeuN for neurons;and GC for oligodendrocytes. Antibodies to tyrosine hydroxylase (TH)were used to identify dopaminergic, noradrenergic, and adrenergicneurons. Dopamine-hydroxylase (DBH) was also used for noradrenergic andadrenergic neurons.

[0098] Astrocytes, neurons, and oligodendrocytes were all found todifferentiate from the MSCs of this invention, indicating that the cellswere multipotent. We also cultured MSCs from transgenic mice whichexpress β-galactosidase off of the neuron specific T1 α-tubulinpromoter, which allowed us to use staining with the ligand X-gal orantibodies for β-galactosidase as an additional neuronal marker. Weobserved β-galactosidase-positive cells.

[0099] Since the majority of differentiated cells remained in clusters,it was not possible to determine the percentage of cells expressing eachmarker. The majority of cells that migrated out of the clusters wereGFAP positive, while a large number of cells were either NeuN orβ-galactosidase positive. A lower number of cells were GC positive.Therefore the MSCs could differentiate into neurons, astrocytes andoligodendrocytes. TH-positive cells were also identified. TheseTH-positive cells are most likely dopaminergic neurons and notnoradrenergic or adrenergic neurons, since no cells were found to be DBHpositive. Significantly, no TH, GFAP or GC positive cells have ever beenreported in vivo in the nasal epithelium. Therefore the olfactoryepithelium-derived nestin-positive MSCs are capable of differentiatinginto cell types (e.g., oligodendrocytes, astrocytes, GABAergic neurons,and dopaminergic neurons) never found in the olfactory epithelium.

[0100] Like the originally-purified olfactory MSCs, MSCs passaged fromtwo to twenty times could also differentiate into neurons, astrocytes,and oligodendrocytes. MSCs which had been passaged were plated onpolylysine-coated dishes. Cells migrated from the clusters and spreadout over the surface of the dish. After 16 DIV, cells that wereimmunopositive for GC, GFAP, βIII tubulin, NeuN, lacZ, or TH could beidentified. Moreover, the proportion of cells positive for the variousmarkers was similar to that seen in the differentiated cultures from theoriginal cultures.

EXAMPLE 3

[0101] Purification of MSCs from Olfactory Epithelial Tissue of AdultMice and Rats

[0102] Similar to the foregoing results, MSCs were also purified fromadult mouse and rat olfactory epithelium and vomeronasal organ using themethods described in Examples 1 and 2.

[0103] Adult mice and rats were anaesthetized with an overdose ofsomnitol, and then decapitated. The olfactory and vomeronasal organepithelia were stripped from the conchae and nasal septum and incubatedin DMEM/F12 medium for one to two days after their dissection and priorto the rest of the purification procedure. After this incubation, theepithelia were dissociated in an identical manner as the epithelia fromjuvenile mice. Two days after the isolation, the majority of the cellswere dead with the exception of a very few large phase bright cells.These cells divided over the next few days, forming small clusters ofdividing cells similar to those described in Example 1. These smallclusters grew to give rise to the large clusters that detached from theculture dish surface. After approximately six divisions, cells in someof these clusters began to differentiate and spread out over the flask'ssurface, while some other clusters, which had been floating, reattachedto the surface and then produced differentiated cells. In some cases,cells multiplied to produce small clusters of cells, but did not grow toform large cell clusters like the postnatal cultures. We have passagedthese cells twenty times using the same procedure as that describedabove with respect to the cells purified from juvenile olfactoryepithelium. These proliferating cells from the adult were alsonestin-positive.

[0104] After the cell clusters derived from adult tissue had beengenerated, the cells could be differentiated into neurons, astrocytes,and oligodendrocytes. Seven days after isolation, clusters were platedonto polylysine-coated 35 mm culture dishes or multi-well culturedishes, in medium containing 2% fetal bovine serum and 2% B-27, but noEGF. Over the next month, cells migrated from the cell clusters and ontothe dish surface. We determined the phenotype of these cells usingantibodies to astrocytes, neurons, dopaminergic neurons, andoligodendrocytes as described above.

[0105] Neurons (including dopaminergic neurons), astrocytes, andoligodendrocytes were found, although the number of these cells was muchlower than the number obtained from the juvenile. The cells purifiedfrom adult olfactory epithelia are self-renewing and multipotent, andthus are MSCs.

EXAMPLE 4

[0106] Purification of MSCs from Mouse Tongue

[0107] We derived MSCs from the tongue, another peripheral tissue thatcontains sensory receptors. The tongue was dissected to remove theepithelial layer that contains the sensory receptors and theirunderlying basal cells. This layer of tissue was triturated to producesingle cells and the single cells were plated in flasks containingDMEM/F12 media supplemented with B-27 and EGF, TGF, and/or bFGF, asdescribed for the olfactory epithelium. After two to three days in a 37°C., 5% CO₂ tissue culture incubator, greater than 99% of the cells inthe culture were dead or dying. A small number (less than 1%) of largephase-bright cells were present, however, most of which attached to theflask bottom. Over the next two to six days, these cells divided andproduced spherical clusters that became larger over time and detachedfrom the flask surface. The cells in these clusters werenestin-positive.

[0108] These nestin-positive MSCs can be passaged using the sametechniques as used for the multipotent stem cells derived from theolfactory epithelium. Similarly, the MSCs can be differentiated intoneurons, astrocytes and oligodendrocytes using the techniques describedherein.

EXAMPLE 5

[0109] Purification of MSCs from Mouse Skin

[0110] Skin from neonatal mice aged 3-15 days was dissociated andcultured in uncoated flasks containing 20 mg/mL EGF and 40 mg/mL bFGF.Over the subsequent one to five days, many (>90%) of the cells die. Asmall population of cells hypertrophy and proliferate to form small cellclusters growing in suspension. Some of these cells first attach to thetissue cluster plastic, hypertrophy and proliferate, and then detach asthe clusters become of sufficient size. Other cells never attach to thetissue culture plastic and instead proliferate in suspension from thebeginning. After four to five days, the cell clusters are small buteasily distinguishable as clusters of non-adherent, proliferating cells.By seven to ten days, many of the cell clusters reach diameters of asmuch as 100 μm, while by two weeks, the cell clusters are macroscopic ifleft unperturbed. Many cells adhered to the plastic, and many died, butby about three to seven days, suspended, non-adherent clusters of up toabout 20 cells formed. These suspended or floating cells weretransferred to a new flask seven days after initial culturing; again,many cells adhered, but the cells in the floating clusters proliferatedto generate larger clusters of more than about 100 cells (FIG. 1A, toppanel). These larger clusters were then isolated, dissociated andpassaged. By this process of selective adhesion, substantially purepopulations of floating clusters were obtained after 3 to 4 weeks. Cellsthat generated these clusters were relatively abundant; 1.5 to 2 cm² ofabdomen skin was sufficient to generate six 25 cm² flasks of floatingclusters over this period of time.

[0111] To determine whether clusters contained MSCs, we dissociated theclusters and plated the cells onto poly-D-lysine/laminin-coated dishesor chamber slides without growth factors and, 12 to 24 hours later,immunostained them for the presence of the neural precursor-specificmarker nestin. After three passages, the majority of the cells expressednestin (FIG. 1B, top panel), a property they maintained over subsequentpassages. They did not, however, express the p75 neurotrophin receptor,a marker for neural crest stem cells, as detected either byimmunocytochemistry or western blots. Additionally, they are negative,as detected by immunocytochemistry, for two proteins characteristic ofmesenchymal stem cells: vimentin and cytokeratin.

[0112] We also determined whether the skin-derived MSCs expressedfibronectin. Four lines of skin-derived MSCs cultured from either adult(FIG. 2; left two columns) or neonatal (right two columns) mouse skin,cultured for either long term (first and third columns) or short term(second and fourth columns) were each dissociated, plated for two daysin DMEM/F12 (3:1) containing 2% B-27 supplement, and then immunostainedfor nestin and fibronectin. As is demonstrated in FIG. 2, the majorityof cells expressed both markers.

[0113] To determine whether clusters of cells could be generated fromadults, skin of adult mice was dissociated and cultured as describedabove. Similar to neonatal mouse skin, most cells adhered to the flaskor died when first cultured. After three to seven days, however,clusters of up to approximately 20 cells were observed that subsequentlyincreased in size. When these cells were passaged at least three times(FIG. 1A, bottom panel), and plated onto poly-D-lysine/laminin overnightin the absence of growth factors, they too were immunopositive fornestin (FIG. 1B, bottom panel) and fibronectin (FIG. 2). Thenestin-positive cells from adults and neonates have been passaged inthis manner for over 30 passages, during which time the number wouldhave theoretically expanded at least 10⁹-fold (assuming a doubling timeof approximately one week).

[0114] To determine whether these nestin-positive, fibronectin-positivecells from skin could generate neural cell types, we analyzed neonatalskin-derived cells after three or more passages and greater by platingthem on poly-D-lysine/laminin in the absence of growth factors.Immunostaining (FIGS. 1C and 1D) and western blot analysis (FIG. 3A)revealed that the skin-derived cells expressed neuronal markers. Atseven days, a subpopulation of morphologically-complex cells coexpressednestin and neuron-specific βIII-tubulin, a profile typical of newly-bornneurons (FIG. 1C). At later time points of 7-21 days, cells alsoexpressed neurofilament-M (NF-M)(FIGS. 1D, 3A), neuron-specific enolase,and NeuN, three other neuron-specific proteins. Finally, someneurofilament-positive cells expressed GAD (FIG. 1D), a marker forGABAergic neurons, which are not found in the PNS. Similar results wereobtained for adult skin-derived MSCs, although at early passages some ofthe βIII-tubulin and neurofilament-positive cells were less typicallyneuronal in morphology.

[0115] Immunostaining and western blots revealed that both neonatal andadult MSCs generated cells expressing the glial markers GFAP and CNPaseat seven to twenty-one days after plating (FIGS. 1D-1F, 2A).Double-labeling for these proteins demonstrated the presence of (i)cells that were GFAP-positive but not CNPase-positive (potentiallyastrocytes), (ii) cells that expressed CNPase but not GFAP (potentiallyoligodendrocytes or their precursors), and (iii) a small subpopulationthat were bipolar and expressed both CNPase and GFAP (potentiallySchwann cells) (FIG. 1E). A subpopulation of GFAP-positive cells alsoexpressed nestin, a finding previously reported for developing CNSastrocytes. Additionally, some cells were positive for A2B5, a markerfor oligodendrocyte precursors (FIG. 4)). Like GAD-positive neurons,astrocytes and oligodendrocytes are normally found only in the CNS.

[0116] Double-labeling studies supported the following additionalconclusions. First, glial versus neuronal markers were expressed indistinct subpopulations of MSCs progeny. Second, after twenty passages,skin-derive MSCs were still able to differentiate into neurons and glialcells. Finally, skin-derived MSCs were able to generate smooth musclecells (as determined by both expression of smooth muscle actin (SMA) andmorphology; FIG. 1G), adipocytes (FIGS. 5 and 6), and bone.

EXAMPLE 6

[0117] MSCs Originate from the Dermal Layer of the Skin

[0118] The two major layers of the skin are the epidermis and thedermis. To determine the origin of the skin-derived MSCs, we dissectedand cultured P7, P14, and P18 mouse epidermis and dermis. The two layersof the skin were separated by incubating the skin pieces (1×2 cm²) in0.2% trypsin at 40° C. for about 24-36 hours, or until the dermis couldbe separated from the epidermis. The cells in each layer weredissociated separately and then cultured in DMEM/F12 (3:1) with B-27supplement, EGF (20 ng/mL) and FGF (40 ng/mL). Only the cells derivedfrom the dermis generated cluster of cells similar to those derived fromwhole skin (FIG. 7A). No viable cells were obtained from the epidermis.To characterize the dermis-derived cell clusters, the clusters werecultured for four weeks and then plated onto tissue culture chamberslides coated with poly-D-lysine and laminin. After 24 hours, the cellswere then processed for immunocytochemistry. Like MSCs derived fromwhole mouse skin, the dermis-derived cells coexpressed nestin andfibronectin (FIG. 7B).

EXAMPLE 7

[0119] Clonal Analysis Indicates that Skin-derived MSCs are Multipotent

[0120] To determine whether skin-derived MSCs are multipotent, weisolated single cells by limiting dilution of cells from clusters thatthree months prior had been derived from neonatal mice. We cultured thecells for five weeks in medium from the same culture line and containinggrowth factor, and then differentiated the cells for two weeks in mediumlacking growth factor but containing 3% rat serum. The cells were thenprocessed for immunocytochemistry. As is demonstrated in FIG. 8, singleclones of cells contained NF-M-and CNPase-positive cells (FIG. 8A), andGFAP- and CNPase-positive cells (FIG. 8B).

EXAMPLE 8

[0121] Western Blot Analysis of Skin-derived MSCs

[0122] For western blot analysis of skin-derived MSCs, four cultures(one adult-derived line and three neonate-derived lines) that had beenpassaged from seven to 40 times were analyzed either as clusters orfollowing differentiation by plating in medium containing 1% FBS, B-27supplement, and fungizone for 14 days in 60 mm dishes coated withpoly-D-lysine and laminin. Cell lysates were prepared, and equal amounts(50-100 μg) of protein from each culture were separated on 7.5% or 10%polyacrylamide gels, transferred to membrane, and then probed withanti-nestin monoclonal antibody (1:1000; Chemicon), anti NF-M polyclonalantibody (1:1000; Sigma), anti GFAP polyclonal antibody (1:1000, Dako),or anti fibronectin polyclonal antibody (1:1000; Sigma). As positivecontrols, we used cortical progenitor cells cultured in the presence ofCNTF (which results in astrocytic differentiation) or in the absence ofCNTF (which results in neuronal differentiation) and adult mouse cortex.As negative controls, we used sympathetic neurons and liver. Asillustrated in FIG. 9A, western blotting confirmed the expression ofGFAP and NF-M in cultures differentiated from both adult and neonateskin-derived MSCs. Similarly, FIG. 9B illustrates the expression of bothnestin and fibronectin in adult and neonate skin-derived MSC clusters.

EXAMPLE 9

[0123] MSC Differentiation can be Modulated by Plating Conditions

[0124] As is illustrated above, when clusters of skin-derived MSCs aredissociated and plated in medium containing FGF and EGF, most of thenestin-positive cells become neurofilament-positive. We have found thatwhen the cells are plated in medium containing 10% FBS, the cells adopta morphology similar to that displayed by adipocytes. The adoption ofthe adipocyte cell fate was confirmed by staining with Oil Red O (FIG.5). The ability of 10% FBS to induce adipocyte differentiation was truefor both adult and neonate skin-derived MSCs (FIG. 6).

EXAMPLE 10

[0125] Pharmacological Inhibitors Affect Survival and Proliferation ofSkin-derived MSCs

[0126] When skin-derived MSCs are plated for three days in proliferationmedium containing FGF, they typically exhibit a spherical morphologycharacteristic of their proliferative state (FIG. 10). We tested theability of pharmacological agents to alter this phenotype. Supplementingthe medium with PD098059 (an inhibitor of the ERK MAPK pathway) causedproliferating cells to flatten and differentiate (FIG. 10), whilesupplementing with LY294002 (an inhibitor of the PI-3-K pathway), causedthe cells to die (FIG. 10). The p38 MAPK inhibitor SB203580 had noobserved effect on the proliferating skin-derived MSCs.

EXAMPLE 11

[0127] Purification of Nestin-positive Cells from Adult Human Skin

[0128] We have purified nestin-positive cells from human scalp. Topurify MSCs from human skin, we utilized tags of scalp tissue generatedby placement of a stereotactic apparatus during neurosurgery. Scalp tagstotalling 1 cm² or less from each of eight individuals were used. Theskin included dermal and epidermal tissue. Tissue was cut into smallerpieces that were then transferred into HBSS containing 0.1% trypsin forforty minutes at 37° C. Following trypsinization, tissue samples werewashed twice with HBSS and once with DMEM:F12 (3:1) supplemented with10% rat serum to inactivate the trypsin. Trypsinized tissue was thenmechanically dissociated by trituration in a pipette and the resultingdispersed cell suspension was poured through a 40 μm cell strainer intoa 15 mL tube. The tube was then centrifuged for five minutes at 1000 rpm(˜1200×g). The cells were resuspended in DMEM:F12 medium containing 40ng/mL bFGF, 20 ng/mL EGF, 2% B-27 supplement, and antibacterial andantifungal agents, and then cultured in 12 well plastic tissue cultureplates. Every seven days, the cell clusters are harvested bycentrifugation, triturated with a fire-polished pasteur pipette, andcultured in fresh medium.

[0129] As for the use of rodent skin, most cells (>75%) adhered to theplastic or died, but after seven days, small floating clusters of cellswere observed. These clusters were then partially dissociated andtransferred to new wells, where they slowly increased in size. Afteradditional passaging, clusters were plated on poly-D-lysine/laminin in3% FBS with no growth factors, and analyzed for the presence of neuralmarkers.

[0130] Within two weeks, greater than 30% of the cells within the cellclusters were nestin-positive (FIG. 3B). Immunolabeling of four to sixweek old cultures also revealed that many of the cells in the clusterswere nestin-positive with the percentage varying from less than 50% togreater than 80% two to three days after plating, and that greater than70% of the cells were fibronectin positive. Double-labelimmunocytochemistry at the same or longer time-points revealed that, inall cultures, some nestin-positive cells also expressed βIII-tubulin anddisplayed elongated neurites (FIG. 3C). Thus, adult human skin is asource for nestin-positive and fibronectin positive MSCs cells that,when differentiated, can express neuron-specific proteins.

EXAMPLE 12

[0131] Purification and Differentiation of MSCs Derived from Other HumanPeripheral Tissues Containing Sensory Receptors

[0132] MSCs can be purified from human olfactory epithelium using thesame procedures as described for the purification of stem cells fromrodent olfactory epithelium. Source material is acquired by surgicalremoval of olfactory epithelial tissue from the donor. Because the MSCsare capable of proliferation and self-renewal, little source tissue isrequired. Preferably, the amount is at least about 1 mm³. Conditions forculturing human cells are described in Example 6, above. Otherconditions are known to those skilled in the art, and can be optimizedfor proliferation or differentiation of neural stem cells, if desired.

[0133] We can purify MSCs from other peripheral tissues containingsensory receptors, other than the olfactory epithelium, tongue, andskin, using techniques described herein. Passaging and differentiationof these cells is also performed using the same techniques describedherein. Other peripheral tissues containing sensory receptors include,for example, mucosal membranes from the mouth or reproductive system.

EXAMPLE 13

[0134] Transformation of MSCs

[0135] In therapy for neurodegenerative diseases, it may be desirable totransplant cells that are genetically modified to survive the insultsthat caused the original neurons to die. In addition, MSCs may be usedto deliver therapeutic proteins into the brain of patients withneurodegenerative disorders to prevent death of host cells. Examplarytherapeutic proteins are described herein. In still another example,MSCs can be induced to differentiate into a desired cell type bytransfecting the cells with nucleic acid molecules encoding proteinsthat regulate cell fate decisions (e.g., transcription factors such asIs1-1, en-1, en-2 and nurr-1, implicated in regulating motorneuron andstriatal phenotypes). Using such a method, it is possible to induce thedifferentiation of the specific cell types required for transplanttherapy. Therefore, it would be advantageous to transfect MSCs withnucleic acid molecules encoding desired proteins. We have previouslyused recombinant adenovirus to manipulate both postmitotic sympatheticneurons and cortical progenitor cells, with no cytotoxic effects. We nowhave established that olfactory epithelial-derived MSCs and skin-derivedMSCs can each be successfully transfected with high efficiency and lowtoxicity. MSCs can be transfected either transiently or stably using notonly adenoviral mediated methods, but also using lipofectamine orelectroporation.

EXAMPLE 14

[0136] Differentiation of MSCs into the Appropriate Cell Type in vivoFollowing Transplantion into Adult Rodent Brain

[0137] One therapeutic use for the MSCs of the present invention isautologous transplantation into the injured or degenerating CNS or PNSto replace lost cell types and/or to express therapeutic molecules. Wedemonstrate below that the MSCs can differentiate into neurons whentransplanted into the adult brain.

[0138] If desired, the dopaminergic innervation of the adult striatumcan be unilaterally destroyed by a local infusion of 6-hydroxydopamineunder conditions in which noradrenergic neurons are spared. Severalweeks later, MSCs are transplanted into both the intact and lesionedstriatum. Altenatively, the cells can be transplanted into unlesionedanimals. The fate of the transplanted MSCs is then determined byimmunohistochemistry. Exemplary transplantation studies are describedbelow. These studies demonstrate that transplanted MSCs candifferentiate into neurons in vivo, as they can in vitro. In the formercase, differentiation and cell fate choice is controlled by the localenvironment into which each cell is placed. Both in vitro-differentiatedand undifferentiated cells are useful therapeutically in the treatment,for example, of neurodegenerative disease (e.g., Parkinson's disease andmultiple sclerosis) or spinal cord injury. For example, dopamingericneurons differentiated from MSCs, or the MSCs themselves, may betransplanted into the substantia nigra or the striatum of patientshaving Parkinson's disease. If desired, the MSCs may also begenetically-modified to express a desired protein.

[0139] In one example, the dopaminergic innervation to adult ratstriatum was first unilaterally lesioned with the chemotoxin6-hydroxydopamine, and the efficacy of the lesions was tested two weekslater by amphetamine-induced rotational behavior. Two days prior totransplantation, rats were immunosuppressed with cyclosporin. MSCs,produced from olfactory epithelia as described herein, were thenstereotactically injected into the caudate-putamen complex on both thelesioned and unlesioned sides. Sixteen days following transplantation,animals were sacrificed, and sections of the striatum were analyzed fornestin- and TH-immunoreactivity. Five of eight animals receivedsuccessful injections of MSCs in the striatum. Of these, four animalsshowed evidence of a nestin-positive tract on both the lesioned andunlesioned sides, although tracts on the lesioned side appeared to bemore intensely nestin-immunoreactive. On adiacent sections, TH-positivecells were observed confined to an area close to the transplant tract onboth the lesioned and unlesioned side. As many as 25-30 TH-positivecells were identified on each section. Cell morphology varied fromsmall, round cells lacking processes to neurons that weremorphologically complex with multiple fine processes. In some cases, theprocesses of these TH-positive neurons extended into the striatum fordistances of up to 300 μm.

[0140] To confirm that these TH-positive neurons derived from the MSCs,we performed two sets of experiments in which the transplanted cellswere detectably-labeled. In one set of experiments, transplanted MSCswere derived from T 1:nlacZ transgenic mice, in which theneuron-specific T1 (α-tubulin promoter drives expression of anuclear-localized β-galactosidase marker gene. Immunohistochemicalanalysis of animals receiving the transgenic MSCs revealed the presenceof B-galactosidase-positive neurons within the transplant tract,confirming that the transplanted MSCs generated neurons in vivo, as theydid in vitro. In a second set of experiments, MSCs were labelled withBrdU for 18 hours, washed to remove the BrdU label, and thentransplanted unilaterally into the 6-hydroxydopamine-lesioned striatumof animals (10 rats, 4 mice) prepared as described herein.Immunohistochemical analysis with an anti-BrdU antibody revealed thatall animals showed evidence of BrdU-positive transplant tracts.Immunocytochemistry with anti-GFAP revealed that, in both xenografts andallografts, GFAP-positive cells with heterogeneous morphology wereconcentrated at the transplant site, but were also present in moderateamounts over the entire ipsilateral hemisphere, with additionalscattered reactive astrocytes seen in the contralateral hemisphere.GFAP-BrdU double-labelled cells were present mainly within or close tothe transplant tract, and varied in morphology from small, round cellswith only a few processes, to large polygonal or fusiform cells withmultiple processes. Immunohistochemistry with anti-TH revealed thatTH-BrdU double-labeled cells were also present, although these were fewin number relative to GFAP-BrdU positive cells. BrdU-TH double-labeledcells were mainly small to medium-sized without processes, although someexamples of double-labeled cells with processes were found within andadjacent to, the transplant tract. Thus, MSCs generated astrocytes andneurons in vivo, and a subpopulation of the latter were TH-positive.Together, these findings show that peripheral tissue-derived MSCs arecapable of generating cell types that are never found within olfactorytissue, including oligodendrocytes and TH-positive neurons.

[0141] To determine whether skin-derived MSCs also generatedifferentiated neural cell types in vivo, we tagged adult mouseskin-derived MSCs with (i) BrdU, and (ii) a recombinant adenovirusexpressing GFP, and then transplanted them as cell clusters of about 20to about 100 cells into the lateral ventricles of P2 rats.Immunostaining fourteen days later revealed that, in all animalsanalyzed (n=8), transplanted cells had migrated extensively (FIG. 11A).In particular, tagged cells had integrated into the cortex, thehypothalamus and the amygdala in all, and into the hippocampus in two ofthe transplanted brains (FIG. 11A). In the cortex, GFP-positive cellswere located in patches (FIGS. 11A, 11B) or occasionally as single cells(FIG. 11C), including some that had integrated into and adopted themorphology of layer V pyramidal neurons (FIGS. 11B, 11C). These cellshad triangular-shaped soma, and projected a presumptive apical dendritefrom layer V towards layer I, in a manner similar to the endogenouslayer V neurons. That these cells were neurons was demonstrated bydouble-labeling for neuron-specific enolase (FIG. 3D).Immunocytochemical analysis also confirmed that these were transplantedcells, as BrdU-positive cells were present in the same locations asGFP-positive cells in all brains (FIG. 11B).

[0142] In both the amygdala and hippocampus, transplanted cells alsodisplayed neuronal morphology. In the amygdala, GFP and BrdU-positivecells were large, with prominent nuclei, and extensive processes (FIG.11E). In the hippocampus, transplanted cells had integrated into boththe dentate gyrus and pyramidal cell layers, and their morphology wastypical of the endogenous granule and pyramidal cells, respectively(FIGS. 11A, 11E). GFP-positive staining was also seen within themolecular layer. Finally, GFP- and BrdU-positive cells were observed inother locations, such as the hypothalamus, where the morphology of manycells was not typically neuronal.

[0143] Skin-derived MSCs tranplanted into adult rats also survive andintegrate. We labeled adult mouse skin-derived MSCs that had beenpassaged more than thirty times with the nuclear dye 33258, washedextensively, and then injected the cells stereotactically into thebrains of adult rats that were immunosuppressed with cyclosporin. Fourweeks later, we sacrificed the animals by perfusion and processed thebrains for histological examination. Hoeschst-labeled cells were presentin the hippocampus, olfactory bulb, and striatum. From these data, weconclude that the transplanted skin-derived MSCs are capable of survivalfollowing transplantation. Moreover, cells are capable of migrating fromthe site of injection to numerous brain regions.

[0144] Skin-derived MSCs are also capable of survival, migration, andintegration following transplantation into a hemisected adult mousespinal cord. In this example, the cells were injected into the injuredsides of hemisected spinal cords. Eight days later, the animals weresacrificed and the spinal cords processed for histological analysis.Hoechst-labeled cells were present at the site of the initial injection,and had also migrated extensively into the injured spinal cord.

EXAMPLE 15

[0145] Differentiation of Non-neural Cells from MSCs

[0146] In addition to being capable of differentiating as neural cells(i.e., neurons, oligodendrocytes, astrocytes, and Schwann cells), theperipheral tissue-derived MSCs are capable of differentiating asnon-neural cells that are normally not found in the tissue from whichthe cells were derived. For example, we have demonstrated that theskin-derived MSCs can differentiate as smooth muscle cells, bone andadipocytes. It is likely that the cells described herein have evengreater potential. Conditions for the differentiation of the MSCs intosmooth muscle cells is described herein.

[0147] Signals or conditions sufficient for inducing MSCs todifferentiate as other cell types (e.g., lymphocytes, cardiac musclecells, skeletal muscle cells, melanocytes, and pancreatic cells) areknown in the art. For example, unique signals induce neuralcrest-derived stem cells to become melanocytes, cartilage, smooth musclecells, or bone (for review, see LaBonne and Bronner-Fraser, J.Neurobiol., 36:175-189, 1998; Sieber-Blum, Intl. Rev. Cytol. 197:1-33,2000). Conditions for inducing CNS-derived neural stem cells todifferentiate as non-neural cells such as smooth muscle cells, skeletalmuscle cells, hepatocytes, hematopoietic cells, osteocytes, andchondrocytes have similarly been elucidated (Bjornson et al., Science283:534-537, 1999; Tsai and McKay, J. Neurosci. 20:3725-3735, 2000;Keirstead et al., J. Neurosci. 19:7529-7536, 1999; Mujtaba et al., Dev.Biol. 200:1-15, 1998; Clark et al., Science 288:1660-1663, 2000).

[0148] Our recent discovery that MSCs maintain the potential to produceboth neural and non-neural cell types has been accompanied by thediscovery that non-neural stem cells such as bone marrow-derived stemcells (i.e., stromal cells or mesenchymal stem cells) also have thepotential to produce a wide variety of neural and non-neural stem cells(Ferrari et al., Science 279:1528-1530, 1998; Gussoni et al., Nature401:390-394, 1999; Peterson et al., Science 284:1168-1170, 1999; Pereiraet al., Proc. Natl. Acad. Sci. USA 92:4857-4861, 1995; Prockop, Science276:71-74, 1997; Kessler and Byrne, Annu. Rev. Physiol. 61:219-242,1999; Pittenger et al., Science 284:143-147). The conditions under whichthese bone marrow-derived cells differentiate as, for example, skeletalmuscle cells, cardiac muscle cells, hepatocytes, adipocytes, osteocytes,or chrondrocytes are likely to be conditions under which the peripheraltissue-derived MSCs would differentiate similarly. Thus, the peripheraltissue-derived MSCs described herein can be induced to differentiateinto both neural and non-neural cells that are not normally found in thetissue from which the MSCs were derived.

[0149] The foregoing experiments were performed using the followingmethods.

[0150] Skin-derived MSC Culture

[0151] For neonatal (three to 14 days) and adult (two months to oneyear) mice, skin from abdomen and back was carefully dissected free ofother tissue, cut into 2-3 mm³ pieces, washed three times in HBSS, andthen digested with 0.1% trypsin for 40 minutes at 37 C., followed by0.1% DNAase for one minute at room temperature. Tissue pieces were thenwashed twice with HBSS, once with media (DMEM-F12, 3:1, 1 g/mlfungizone, 1% penicillin/streptomycin) containing 10% rat serum (HarlanBioproducts), and twice with serum-free media. Skin pieces were thenmechanically dissociated in media, and the suspension poured through a40 M cell strainer (Falcon). Dissociated cells were centrifuged, andresuspended in 10 ml media containing B-27 supplement, 20 ng/ml EGF and40 ng/ml bFGF (both Collaborative Research). Cells were cultured in 25cm² tissue culture flasks (Corning) in a 37 C., 5% CO₂ tissue cultureincubator.

[0152] To culture human skin-derived MSCs, two to three pieces of scalptissue ranging between 4-9 mm² (generated by placement of thestereotaxic apparatus for neurosurgery) were washed with HBSS, anysubcutaneous tissue was removed, and the skin was cut into small pieces1-2 mm³ in size. Tissue pieces were transferred to 15 mL Falcon tubes,washed three times with HBSS, and enzymatically digested in 0.1% trypsinfor 40 minutes at 37 C., and then washed as for mouse tissue.Dissociated cells were suspended in 5 mL of the same media used formouse cultures, with the addition of 20 ng/ml LIF (R&D Systems Inc.).The cell suspension was placed in Falcon 6-well tissue culture platesand maintained in a 37 C., 5% CO₂ tissue culture incubator. Cells weresubcultured by partial dissociation of the clusters that formed every 7to 10 days.

[0153] To passage floating clusters of cells, the medium containing thecell clusters was centrifuged, the cell pellet mechanically dissociatedwith a fire-polished Pasteur pipette, and the cells reseeded in freshmedia containing B-27 supplement and growth factors as above. Cells werepassaged every 6 to 7 days. For induction of differentiation into smoothmuscle cells, the cell clusters were centrifuged, the growthfactor-containing supernatant removed, and the clusters resuspended infresh media containing B-27 supplement and either 3% rat serum or 1-3%fetal bovine serum. The clusters were then plated onto 4-well Nunclonculture dishes coated with poly-D-lysine/laminin, and the medium waschanged every 3 to 7 days.

[0154] Transplantation of Ofactory Epithelium-derived MSCs

[0155] Olfactory epithelium-derived MSCs were purified and cultured asdescribed herein. Female Sprague-Dawley rats or CD1 albino mice (CharlesRiver, Montreal, Quebec, Canada) weighing 180-200 g or 25-30 g,respectively, were anaesthetized with a mixture of ketamine (90 mg/kg)and xylazine (10 mg/kg) (intraperitoneal) prior to stereotacticinjections of 24 μg of 6-hydroxydopamine hydrobromide (dissolved in 5 μLof 0.9% saline containing 0.2 mg/ml ascorbate) into the right medialforebrain bundle (Tooth bar: −2.4 mm; A: −4.4 mm; L: 1.0 mm; V: 7.5 mm).Two weeks after the lesion, animals were tested for rotational behavior.Animals were immunosuppressed with cyclosporine (40 mg/kg,intraperitoneal) once a day until the day of sacrifice. For MSCtransplantation, anaesthetized animals were mounted in a Kopfstereotactic apparatus, and 2×2.5 μL aliquots of MSCs were injectedunilaterally into the lesioned caudate putamen or bilaterally in someanimals. The injections were made using a 5 μL Hamilton syringe at thefollowing coordinates: Tooth bar, −2.4 mm; A: 0.2; L: 3.0; V: 5.5-6.0.Injections were performed over a period of three minutes, a further fiveminutes was allowed for diffusion, and the needle was then retracted.These 5 μL injections contained MSCs derived from one neonatal pupcultured for 7 to 14 days. For the BrdU experiments, BrdU (10 μM) wasadded to culture media for 18 hours, after which the MSCs were washedthree times with fresh media to remove the BrdU, and then transplantedone day later.

[0156] Transplantation of Skin-derived MSCs

[0157] Labeling of skin-derived MSCs was performed as follows. Threedays prior to transplantation, free-floating cell clusters werepartially dissociated by gentle trituration, and then exposed to 50 MOIof a recombinant adenovirus expressing GFP, using standard techniques.Twenty-four hours later, the MSCs were centrifuged, washed, andresuspended in fresh medium containing 2 μM BrdU for an additional twodays. Prior to transplantation, MSCs were rinsed five times with freshmedium and resuspended to a concentration of 50,000 cells/μl. At thetime of transplantation, approximately 75% of the MSCs expressed GFP,while 95% were BrdU positive.

[0158] MSCs labeled with BrdU and GFP were stereotaxically injected intothe right lateral ventricle of cryoanaesthetized two day old rat pups(co-ordinates from Bregma: lateral 1.5 mm, ventral 3.3 mm).Approximately 50,000 cells were injected over a three minute period in avolume of 1 μL. Fourteen days following transplantation, mice wereperfused with 50 mL 4% formaldehyde buffered with PBS. Fifty microncoronal sections through the forebrain were cut using a freezingmicrotome and analyzed immunocytochemically. All eight animals receivingcell transplants showed extensive labeling for tagged cells. No evidenceof tumor formation was observed.

[0159] Immunostaining

[0160] Immunostaining of olfactory epithelium-derived MSCs was performedas follows. With the exception of GC immunocytochemistry, culture disheswere washed twice with Tris-buffered saline (TBS; 10 mM Tris, 150 mMNaCl, pH 8), then fixed with 4% formaldehyde, washed three times withTBS, blocked with TBS plus 2% goat serum (Jackson ImmunoResearch,Mississuagua, Ontario, Canada), and 0.1% Triton-X (Sigma Chemicals, St.Louis Mo.) for 30 minutes, then incubated with primary antibody in TBSplus 2% goat serum. Following primary antibody incubation, the disheswere washed three times with TBS, incubated in secondary antibody in TBSplus 2% goat serum, washed three times, and then viewed with afluorescence inverted microscope. The antibodies to GFAP (BoehringerMannheim,Laval, Quebec, Canada), βIII tubulin (Sigma), NeuN (Dr. R.Mullen), MAP-2 (clone AP-20; Sigma), and NF-160 (American Tissue CultureCollection, Manassas Va.) were monoclonal antibodies used atconcentrations of 1:200; 1:25; 1:10, and 1:1 respectively. Antibodies tonestin (a gift from Dr. Ron MacKay (National Institutes of Health), TH(Eugenetech Eugene, Oreg.), and DBH (Eugenetech) were rabbit polyclonalantibodies used at concentrations of 1:1000, 1:200, and 1:200respectively. Secondary antibodies Cy3 conjugated goat anti-mouse(Jackson ImmunoResearch) and Cy3 conjugated goat anti-rabbit (JacksonImmunoResearch), and were used at 1:1500. For double-labellingexperiments, we used FITC goat anti-mouse (Jackson ImmunoResearch).

[0161] For GC immunocytochemistry, living cultures were incubated inDMEM containing HEPES, 5% heat inactivated horse serum (HS), and 1:10 GCantibody for 30 min at 37 C., washed three times with themedium/HEPES/HS, fixed with 4% formaldehyde for 15 minutes, rinsed threetimes in TBS, incubated in Cy3 conjugated goat anti-mouse antibody(1:1500) for two hours, and finally washed three times in TBS. Culturesprocessed for immunocytochemistry without primary antibodies revealed nostaining.

[0162] Immunocytochemical analysis of cultured skin-derived MSCs wasperformed as follows. The primary antibodies that were used were:anti-nestin polyclonal (1:250, Dr. Ron McKay, NINDS), anti-nestinmonoclonal (1:400, PharMingen Inc.), anti-βIII-tubulin monoclonal(1:500, Tuj1 clone, BabCo), anti-neurofilament-M polyclonal (1:200,Chemicon Intl.), anti-GAD polyclonal (1:800, Chemicon Intl.), anti-NSEpolyclonal (1:2000, Polysciences Inc.), anti-GFAP polyclonal (1:200,DAKO), anti-CNPase monoclonal (1:400, Promega), anti-p75 NTR polyclonal(1:500, Promega), anti-SMA monoclonal (1:400, Sigma-Aldrich), andanti-A2B5 monoclonal (Dr. Jack Snipes, M.N.I.). The secondary antibodieswere Cy3-conjugated goat anti-mouse (1:200), Cy3-conjugated goatanti-rabbit (1:400), FITC-conjugated goat anti-mouse (1:50-1:100), andFITC-conjugated goat anti-rabbit (1:200) (all from JacksonImmunoresearch Laboratories).

[0163] Immunocytochemical analysis of free-floating brain sections wasperformed by DAB immunohistochemistry. For GFP, sections were incubatedin 0.3% H₂O₂ for one hour to inhibit endogenous tissue peroxidaseactivity prior to blocking. For BrdU immunohistochemistry, sections werepre-incubated in 0.5% sodium borohydride for 20 minutes prior toblocking of endogenous peroxidase activity in 0.03% H₂O₂ for 30 minutes.To permeabilize the nuclei for BrdU immunohistochemistry, sections wereincubated in 1% DMSO for 10 minutes, the DNA denatured with 2N HCl for60 minutes, and the HCl neutralized with 0.1M borate buffer for 5minutes. All sections were blocked for one hour in 10% BSA, and thenincubated for 48 hours at 4° C. with either anti-GFP (1:1000, Clontech)or anti-BrdU (1:100, Becton-Dickinson). Primary antibodies were detectedusing a biotinylated horse anti-mouse secondary antibody (1:200, VectorLaboratories) for one hour at room temperature, and visualized using theVectastain kit (Vector Laboratories) and a nickel-enhanced DAB reactioncontaining 0.05% DAB, 0.04% nickel chloride, and 0.015% H₂O₂. Sectionswere mounted onto slides, dehydrated through a series of ethanols andHistoclear (Fisher Scientific), and coverslipped using Permount (FisherScientific).

[0164] Fluorescence immunohistochemistry was performed to co-localizeGFP expression with NSE. Free-floating sections were blocked in 10% BSAfor one hour at room temperature, and then incubated 48 hours at 4° C.in a solution containing mouse anti-GFP and rabbit anti-NSE. Sectionswere incubated with Cy3 conjugated anti-mouse and FITC conjugatedanti-rabbit secondary antibodies for one hour at room temperature, andcoverslipped using Sigma Mounting Medium.

Other Embodiments

[0165] The present invention has been described in terms of particularembodiments found or proposed by the present inventors to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. All such modifications are intended to beincluded within the scope of the appended claims.

[0166] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

What is claimed is:
 1. A cellular composition comprising a purifiedpopulation of multipotent mammalian cells, which multipotent cells formnon-adherent clusters in culture, are self renewing, are positive fornestin and fibronectin protein, and differentiate into both neuronal andnon-neuronal cell types.
 2. A cellular composition comprisingmultipotent mammalian cells with fewer than 30 percent lineage committedcells, wherein said multipotent cells form non-adherent clusters inculture, are self renewing, are positive for nestin and fibronectinprotein, and differentiate into ectodermal and mesodermal cell types. 3.A cellular composition comprising multipotent cells prepared by themethod comprising: (a) culturing a dissociated sample of epithelialtissue; (b) isolating, from the culture, non-adherent cellscharacterized by the following: are positive for nestin and fibronectinprotein, are self renewing, and differentiate into ectodermal andmesodermal cell types.
 4. A cellular composition comprising a populationof multipotent mammalian cells, which multipotent cells formnon-adherent clusters in culture, are self-renewing, are positive fornestin and fibronectin protein, differentiate into both neuronal andnon-neuronal cell types, and can proliferate in culture in the absenceof exogenous EGF.
 5. A cellular composition comprising a population ofmultipotent mammalian cells, which multipotent cells form non-adherentclusters in culture, are self-renewing, are positive for nestin andfibronectin protein, are negative for vimentin and cytokeratin protein,and differentiate into both neuronal and non-neuronal cell types.
 6. Acellular composition comprising a population of multipotent mammaliancells, which multipotent cells form non-adherent clusters in culture,are self-renewing, are positive for nestin and fibronectin protein, arenegative for vimentin, cytokeratin, and p75 protein, and differentiateinto both neuronal and non-neuronal cell types.
 7. The cellularcomposition of any of claims 1-6, which multipotent cells differentiateinto cells expressing one or more markers selected from the groupconsisting of Glial Fibrillary Acid Protein (GFAP), neurofilament 160,βIII tubulin, NeuN, neurofilament-M (NFM), neuron-specific enolase,galactocerebroside, GAD, tyrosine hydroxylase (TH), dopamineβ-dehyrdogenase and CNPase.
 8. The cellular composition of any of claims1-6, which multipotent cells can differentiate to produce astrocytes,oligodendrocytes, and neurons.
 9. The cellular composition of claim 8,wherein the neuron is a dopaminergic neuron.
 10. The cellularcomposition of any of claims 1-6, which multipotent cells differentiateto form cells selected from the group consisting of epithelial cells,endothelial cells, skeletal muscle cells, cardiac muscle cells,connective tissue cells, lung cells, adipocytes, pancreatic islet cells,hematopoietic cells, chondrocytes, bone, kidney cells, and hepatocytes.11. The cellular composition of any of claims 1-6, wherein themultipotent cells are present in the composition in non-adherent cellaggregates of 10 cells or more.
 12. The cellular composition of any ofclaims 1-6, including 10⁶ or more of said multipotent cells.
 13. Thecellular composition of any of claims 1-6, wherein the multipotent cellsare engineered to include a heterologous gene.
 14. The cellularcomposition of claim 13, wherein the heterologous gene encodes atherapeutic protein.
 15. The cellular composition of claim 13, whereinthe heterologous gene encodes a protein which affects the growth ordifferentiation fate of said multipotent cells or the progeny thereof.16. The cellular composition of claim 3, wherein said epithelial tissueis skin or mucosal tissue.
 17. The cellular composition of claim 3,wherein said epithelial tissue is derived from tongue.
 18. The cellularcomposition of claims 3, 16 or 17, wherein said epithelial tissue isfrom an adult mammal.
 19. The cellular composition of claims 3, 16 or17, wherein said epithelial tissue is from a juvenile mammal.
 20. Thecellular composition of the differentiated cells of any of claims 1-6.21. A cellular composition comprising differentiated cells prepared bythe method comprising plating the non-adherent clusters of any of claims1-6 on a substratum coated with a substrate that promotes theirattachment to the substratum.
 22. A kit comprising a cellularcomposition of any of claims 1-6, and means for introducing the cellularcomposition into a patient.
 23. A kit comprising a composition ofdifferentiated progeny from a multipotent cell of any of claims 1-6, andmeans for introducing the composition into a patient.
 24. The cellularcomposition comprising any of claims 1-6, formulated in apharmaceutically acceptable carrier, auxiliary or excipient.
 25. Thecellular composition comprising claim 20, formulated in apharmaceutically acceptable carrier, auxiliary or excipient.
 26. Thecell line established from the cellular composition of any of claims1-6.
 27. The cell line of claim 26, wherein the cell expresses aheterologous gene.
 28. A method of treating a patient with cell damageor disease comprising transplanting the cells of any of claims 1-6. 29.The method of claim 28, wherein the multipotent cells are autologouslyderived.
 30. The method of claim 28, wherein the multipotent cells arederived from a genetically related donor.
 31. The method of claim 28,wherein the cell damage or disease is selected from a neurodegenerativedisease, diabetes, heart disease, heart attack, or stroke.
 32. Themethod of claim 28, wherein the cell damage or disease is the resultbacterial or viral infection.
 33. The method of claim 28, wherein thecell damage or disease is the result of traumatic injury includingfractures, lacerations, and burns.
 34. The method of claim 28, whereinthe multipotent cells are transplanted at the site of cell damage ordisease.
 35. The method of claim 28, wherein the multipotent cells aredelivered to the site of cell damage via the bloodstream.
 36. The methodof claim 28, wherein the patient is a human patient.
 37. A cellularcomposition comprising a purified preparation of the differentiatedcells of claims 20 or
 21. 38. A method of treating a patient with celldamage or disease comprising transplanting the cells of claim
 37. 39.The method of claim 38, wherein the patient is a human patient.
 40. Themethod of claim 38, wherein the transplant is at the site of cell damageor disease.
 41. The method of claim 38, wherein the cell damage ordisease is a neurodegenerative disease, diabetes, heart disease, heartattack, or stroke.
 42. The method of claim 38, wherein the cell damageor disease is the result bacterial or viral infection.
 43. The method ofclaim 38, wherein the cell damage or disease is the result of traumaticinjury including fractures, lacerations, and burns.
 44. A method forpreparing stem cell preparations, comprising: (a) obtaining anepithelial tissue sample from a patient; (b) culturing cells dissociatedfrom said tissue sample; (c) isolating from the culture multipotentcells characterized by the following: form non-adherent clusters inculture; are self renewing; express nestin and fibronectin; anddifferentiate into ectodermal and mesodermal cell types; and (d)preserving and storing the multipotent cells for later retrieval.
 45. Amethod for preparing cell preparations, comprising: (a) obtaining anepithelial tissue sample from a patient; (b) culturing cells dissociatedfrom said tissue sample under conditions wherein multipotent cells areexpanded, which multipotent cells are characterized by the following:form non-adherent clusters in culture; are self renewing; express nestinand fibronectin; and differentiate into ectodermal and mesodermal celltypes; (c) differentiating the multipotent cells into one or morelineage committed cell types; and (d) preserving and storing thedifferentiated cells for later retrieval.
 46. The method of either claim44 or 45, wherein the preserved cells are formulated in apharmaceutically acceptable carrier, auxiliary or excipient.
 47. Themethod of either claim 44 or 45, wherein the step of preserving themultipotent cells or differentiated cells includes cryogenicpreservation.
 48. A method for conducting a regenerative medicinebusiness, comprising: (a) a service for accepting and logging inepithelial tissue samples from a client; (b) a cell culture system forculturing cells dissociated from said tissue sample, which systemprovides conditions suitable for expanding multipotent cells in saidsample, which multipotent cells are characterized by the following: formnon-adherent clusters in culture; are self renewing; express nestin andfibronectin; and differentiate into ectodermal and mesodermal celltypes; (c) a cell preservation system for preserving said multipotentcells for later retrieval on behalf of said client or other third party.49. A method for conducting a regenerative medicine business,comprising: (a) a service for accepting and logging in epithelial tissuesamples from a client; (b) a cell culture system for culturing cellsdissociated from said tissue sample, which system provides conditionssuitable for expanding multipotent cells in said sample, whichmultipotent cells are characterized by the following: form non-adherentclusters in culture; are self renewing; express nestin and fibronectin;and differentiate into ectodermal and mesodermal cell types; (c) a celldifferentiation system for differentiating said multipotent cells intoone or more lineage committed cell types (d) a cell preservation systemfor preserving said lineage committed cells for later retrieval onbehalf of said client or other third party.
 50. The method of claim 48or 49, further including a billing system for billing the client or amedical insurance provider thereof.
 51. A method for conducting a stemcell business, comprising: (i) identifying one or more agents whichaffect the proliferation or differentiation of the multipotent cells ofany of claims 1-6; (ii) conducting therapeutic profiling of agentsidentified in step (i), or analogs thereof, for efficacy and toxicity inanimals; and (iii) formulating a pharmaceutical preparation includingone or more agents identified in step (ii) as having an acceptabletherapeutic profile.
 52. The method of claim 51, wherein step (i)comprises contacting the multipotent stem cells with one or more smallorganic molecules and identifying those which affect the proliferationor differentiation of the multipotent stem cells.
 53. The method ofclaim 51, wherein step (i) comprises contacting the multipotent stemcells with one or more extracellular proteins and identifying thosewhich affect the proliferation or differentiation of the multipotentstem cells.
 54. The method of claim 51, including an additional step ofestablishing a distribution system for distributing the pharmaceuticalpreparation for sale.
 55. The method of claim 51 or 54, includingestablishing a sales group for marketing the pharmaceutical preparation.56. A method of conducting a drug discovery business, comprising: (i)identifying one or more agents which affect the proliferation ordifferentiation of the multipotent cells of any of claims 1-6; (ii)licensing, to a third party, the rights for further drug development ofagents identified in step (i) as able to affect the proliferation ordifferentiation of the multipotent stem cells.